JP2001522352A - Antigenic structural peptides, antigenic and immunogenic compounds, and uses for detecting, preventing and treating HCV infection - Google Patents

Abstract

(57) The present invention comprises the N-terminal 2-45 amino acid sequence of the core or nucleocapsid (p21) of hepatitis C virus (HCV) or a mutant of any of the above viruses, including the N-terminus Or a structural peptide identified by an antibody directed against a polypeptide other than a protein or peptide compound consisting thereof. The present invention provides a method wherein the structural peptide comprises a first peptide fragment having a secondary structure in the form of an α helix, a second peptide fragment having a secondary structure in the form of an α helix, and a second peptide fragment which binds two α helices. It comprises three connecting peptide fragments, characterized in that these two α-helices are substantially perpendicular to one another in space. This peptide can be used to detect antibodies directed against the HCV p21 protein, to detect all or a portion of the HCV RNA, and to detect, prevent or treat HCV infection in a diagnostic, prophylactic or therapeutic manner. It can be used for the preparation of a composition.

Description

DETAILED DESCRIPTION OF THE INVENTION             Antigenic structural peptides, antigenic and immunogenic compounds,            And uses for detecting, preventing and treating HCV infection   The present invention relates to hepatitis C virus (HCV) core or nucleocapsid proteins. Immunodominant multimer located at the N-terminus of protein (or p21 protein) Regarding the characterization of antigenic sites belonging to the pitope region [B. Hosejn et al., Proc. Natl. Acad. Sci. USA, 88, 3647-51 (1991)], as well as particularly for the diagnosis, treatment or prevention of HCV infection. And applications derived from the above sites.   According to document EP-A-0 569 309 in the name of the applicant, HCV From the N-terminal 2nd amino acid to the 45th amino acid of the nucleocapsid protein A peptide having a sequence represented by SEQ ID NO: 1 is well known. This Peptides are similar to nucleocapsid proteins, To obtain sensitivity and specificity in terms of the detection of directed antibodies To determine which immunodominant region is sufficient.   Continuing with work on peptides as defined above, Applicants made the following findings: Done:   -N-terminal 15 to 39 amino acids of the nucleocapsid protein The barrel region defines the location of the immunodominant epitope,   This immunodominant epitope has a structural type, Or L-helix type three-dimensional structure, in which case two The ricks are arranged substantially perpendicular to each other.   Therefore, the present invention relates to hepatitis C virus (HCV) or any of the above viruses. Contains the sequence of the N-terminal 2-45 amino acids of the mutant core protein (p21) For structural peptides recognized by antibodies directed against the polypeptide, The peptide has at least a first having a secondary structure in the form of an alpha helix. Peptide fragment, a second peptide fragment having a secondary structure in the form of an alpha helix A fragment and a fourth binding peptide fragment joining the two α helices. Including or consisting of the following structures, these two alpha helices are spatially alternating Is substantially vertical. The angle formed by the two α helices is advantageous Is 90 ° ± 10 °.   The third binding peptide fragment is composed of loops and L-shaped secondary structures, especially from βL-shaped It is preferably selected.   In accordance with the present invention, a total comprising or consisting of the N-terminus of the p21 protein All protein or peptide compounds have been excluded from the definitions above and below. Which is considered to start at the amino acid at position 1 or the amino acid at position 2. .   Advantageously, the peptide sequence of the peptide of the invention is such as SEQ ID NO: 3 to 10 Also selected from any peptide sequence equivalent to SEQ ID NO: 2.   Before describing the invention in more detail and presenting its importance and application, Descriptions of terms used in the claims are set forth below.   Structural epitopes are separated in the protein sequence, but not in the polypeptide chain. Due to the holding, they become spatially close to each other, and Amino acids recognized by antibodies, forming units that can be recognized by Is understood to mean the protein region determined by   Peptide is an amino acid obtained by chemical synthesis or genetic recombination. Shows a small piece of acid. The peptide according to the present invention can be used, for example, using an automatic peptide synthesizer. By traditional synthetic methods or expression such as plasmids or viruses Inserting a DNA sequence encoding the peptide into a vector; and Transform eukaryotic or prokaryotic cells using expression vectors And obtaining them by genetic engineering methods, including culturing these cells. It is possible.   A reference peptide sequence having a reference unit (such as SEQ ID NO: 2 in the present invention) Equivalent peptide sequences are defined as insertions and / or deletions of one or more amino acids and And / or substitution and / or extension and / or shortening and / or chemical modification Is understood to mean an amino acid sequence modified by On the other hand, it substantially preserves or even amplifies the antigenic properties of the reference peptide sequence, On the other hand, it essentially preserves the reference quaternary structure of the reference peptide sequence.   Therefore, "equivalent sequence" means that one or more amino acids is one or more other amino acids. A sequence replaced by: one or more amino acids of the L series is one of the D series Sequences substituted by the above amino acids; acetylation of amine function, thiol Carboxylation of functional groups, esterification of carboxyl functional groups, acid by sulfur atom Sequences in which amino acid side chain modifications such as substitution of elementary atoms have been introduced; Inverse, retro-inverse, reduction and methyleneoxy linkages It is understood that modification of the peptide bond is particularly meant.   A peptide sequence equivalent to SEQ ID NO: 2 is also a peptide completely different from SEQ ID NO: 2 While having the same antigenic properties and potentially similar quaternary structure as SEQ ID NO: 2. Includes those with mimotope peptides.   A peptide sequence equivalent to SEQ ID NO: 2 also differs little from SEQ ID NO: 2 While having a peptide sequence, a quaternary structure similar to SEQ ID NO: 2 and similar antigenicity Includes those with quality mimotope peptides. These mimotope peptides are , By way of example, by the peptide sequence of SEQ ID NOs: 3 to 10.   The secondary structure of a peptide fragment is determined by hydrogen bonds containing the atoms that form the primary structure. It is essentially defined as a stabilized three-dimensional structure. Some ties of secondary structure And, in particular, a structure in the form of an α-helix and a structure in the form of a βL may be distinguished .   α helix, Pauling et al. (L. Pauling, Proc. Natl. Acad. Sci. USA, 37, 205-211 ( 1951)] and corresponds to the right helix. The α helix is oxygen of the carbonyl group of the amino acid at position i and hydrogen of the amide group of the amino acid at position i + 4 And is essentially stabilized by hydrogen bonds between These hydrogen bonds form the helicopter. N, H and O involved in hydrogen bonding are actually the same On the line. 1.5 ° apart from the next amino acid residue on the axis of the α helix, One complete turn corresponds to 3.6 amino acid residues. Movement along the helix axis Therefore it is 5.4 ° per revolution.   The parameters of the alpha helical structure are:   Angle expressed in degrees:   Φ-57   ψ -47   ω 180   Amino acids per turn 3.6   A (C_αi-C_{α + 1}) Distance between residues 1.5   The βL shape is considered as a secondary structure in which at least seven different types are described. [P.Chou and G.D.Fasman, J. Mol. Biol., 115, 135-175 (1977)]. They are Allows polypeptide chain folding and contains a minimum of 4 amino acids. They are It generally allows for a change in orientation between the two secondary structures.   Finally, a loop not generally considered as a secondary structure is the Ω A polypeptide chain that describes a letter very frequently and therefore has the name omega loop (J. Leszczinski and J. Rose, Sciences, 234, 849-855 ( 1986]). A loop is defined by its length and the short distance between the ends. loop Is defined and stabilized by interactions at the level of the side chains of its constituent residues.   In order to obtain a unit or a reference quaternary structure corresponding to the above definition according to the present invention, It is the nucleocapsid protein of hepatitis C virus, or a modification of the virus. From the N-terminal 15th amino acid to the 39th amino acid of the variant nucleocapsid protein It preferably contains a primary structure equivalent to amino acids spanning amino acids.   More advantageously, the primary structure is any peptide sequence equivalent to SEQ ID NO: 2 Or consist of them. The structural peptide is identified by SEQ ID NO: 2 The first peptide having a secondary structure in the form of an α helix Fragment is Thr Asn Arg Arg Pro Gln Asp Val Lys  Consists of a Phe sequence (1 to 10) and has a secondary structure in the form of an α helix The second peptide fragment was Gln Ile Val Gly Gly Val Tyr. A third peptide consisting of a Leu Leu Pro Arg sequence (15 to 25); The fragment consists of the Pro Gly Gly Gly sequence (11 to 14).   The invention also relates to hepatitis C virus (HCV) or any of the above viruses. Comprising the sequence of the N-terminal 2-45 amino acids of the mutant core protein (p21); Or recognized by an antibody directed against a polypeptide consisting thereof Antigenic compounds as well as immunogenic compounds capable of inducing the production of antibodies The antigenic or immunogenic compound comprises a peptide of the invention and comprises a p21 protein K Any protein or peptide compound comprising or consisting of a quality N-terminus This terminus begins with an amino acid at position 1 or an amino acid at position 2 Will be considered. By way of example, the peptides according to the invention can be prepared by methods known to those skilled in the art. According to, for example, natural or recombinant proteins, synthetic polymers of amino acids Such carrier molecules, or aliphatic chains, nucleic acid fragments or tracers or matrices. And the tracer or marker molecule may be, for example, an oligonucleotide. Leotide or horseradish peroxidase, alkaline phosphatase Or an enzyme such as β-galactosidase, or alternatively, a radioactive element. And so on. The peptide of the present invention is attached to any of the supports. It is.   For example, using the immunogenic compounds described above under traditional physicochemical conditions Monoclones obtainable by immunizing animals or immunizing in vitro Local or polyclonal antibodies also form a subject of the present invention.   The subject of the invention described above, mainly antigenic or immunogenic compounds and pepti Is directed against p21 of HCV or a variant of any of the above viruses Antibodies described above, which can be used to detect and / or quantify Is used to detect and / or determine the antigen of HCV or a variant of any of the above viruses. Used to weigh.   Further, these subjects include infection of HCV or a variant of any of the above viruses. Diagnostic, prophylactic or therapeutic composition intended to detect, prevent or treat Applied for the preparation of products.   Accordingly, the present invention also relates to the units, peptides, compounds and / or Or a pharmaceutical composition comprising the antibody.   The invention further relates to:   -Any of the peptides and / or compounds described above, or any of the antibodies of the invention. Contact the sample, between the antibody and the peptide or compound, or Detecting the formation of any immune complex between the antigen and the antibody of the invention. HCV nuclei of HCV or a variant of said virus in a sample comprising the steps of Detect and detect either leocapsid protein or antibodies directed against the antigen And / or a method of quantification;   Contacting a sample with a peptide and / or compound of the invention and applying HCV R Detecting the formation of a complex between NA and said peptide or compound Detecting and / or determining all or a portion of HCV RNA in a sample, including How to weigh.   Furthermore, the present invention provides a conjugate comprising the peptide of the present invention, For molecular structures that bind to, for example, peptide fragments, nucleotide fragments , And chemical molecules such as those of the functionalized aromatic type. Will Complexation of the peptide of the present invention, which exhibits high affinity with the RNA, is dependent on the adhesion and And the latter can be particularly prevented. Therefore, this complex is Applicable in therapy.   The invention and its features are elaborated in the following examples and in FIGS. You.   FIG. 1 shows a 40% mixture of 2,2,2-trifluoroethanol (TFE) (v / V) / 60% buffer: 100 mM NaCl, 10 mM sodium phosphate Of the three-dimensional structure of the peptide of SEQ ID NO: 2 in solution at pH 5.9 FIG. 2 is a schematic illustration with the help of pt software.   FIGS. 2 and 3 show the N of the peptide capsid (15-41) discussed in Example 3. 1 shows an MR spectrum.   FIG. 4 represents the superposition of the eight structures between Arg18 and Phe24. The RMSD of the main chain between these residues is 0.29 °.   FIG. 5 shows a superposition between Glu29 and Leu37 having the same structure of FIG. The RMSD of the main chain between these residues is 0.39 °.   FIGS. 6 and 7 show Arg18 and Leu37, represented under two different angles. Corresponds to the superposition of the structure of 8 between; the main chain between these residues The RMSD is 0.64 °.   4 to 7 are discussed in Example 4. Example 1 Structural Analysis of Peptide SEQ ID NO: 2 by NMR and Molecular Modeling a) Sequence identity search   The peptides studied have already been determined and the PDB databank (Protein D ata Bank) does not have sequence identity with the conserved protein An attempt was made to find out. Two polypeptide sequences are greater than 30% of each other Identities, they generally have a very similar three-dimensional structure. Conceivable. However, using the search software FASTA 2.03 These analyzes performed by [D.J.Lipman and W.R.Pearson, Sciences, 227, 143 5-1440 (1985)], considering molecular modeling based on identity, This indicates that there is no sequence of a known three-dimensional structure that is homologous to the protein. b) Conditions for solvating the peptide   The peptide was purified from 2,2,2-trifluoroethanol (TFE) at 40% / H_Two O, 0.1 M NaCl, pH 10 at 60C in 60% 10 mM sodium phosphate Analyzed by placing at 5.9. These conditions affect the secondary structure of the peptide. While stabilizing, it is possible to preserve its quaternary structure. c) Conditions for acquisition of NMR spectrum   All NMR experiments were performed on a spectrometer equipped with ultrasim AN Unity +, 500 MHz, 5 mm proton-carbon-nitrogen with Z gradient Performed with the help of a triple rotating probe. Transfer data to SUN IPX Work VNMR software on the station (version 5.1, VARIAN Recorded with the help of). The pulse sequence used was C OSY, TOCSY, NOESY and ROESY. NMR experiments were performed using St aes-TPPI method [D. Marion et al., J. Magn. Res., 85, 393-399 (1989)]. And recorded in phased mode.   2048 data points at dimension t2 and 512 at t1 Run the experiment in increments, with a spectral width of 5500 Hz in both dimensions. And each increment accumulated 32 or 64 times. About the DQF-COSY experiment, 4 K data points and 512 increments were recorded. During the 1.5 second interval relaxation time (and Force pre-saturation at the frequency of water applied during the mixing time in the NOESY and NOESY experiments) Was commonly used to eliminate solvent resonances. About the TOCSY experiment, The isotropic mixture gives a delay Δ equal to 2.6 times the pulse duration at 90 ° to the pulse. MLEV-17 sequence [clean-TOCSY; C. Grie Singer et al., J. Am. Chem. Soc., 110, 7870-7872 (1988)]. 2ms trim-pulss e precedes the mixing time, whose duration is typically set to 80 ms. ROS For the EY experiment, the spin-lock has a power output between 3 and 4 kHz. Achieved by continuous radiation. Two pulses at 90 ° are on both sides of the mixing time (Equilibrium ROSEY). d) Molecular modeling   All molecular modeling steps were performed using the IBM rs6000 model 560 or SUN X-lor program version 3.1 on Spark20 station [A.T.Brunger, in X-plor, A System fo r Crystallography and NMR, Yale University Press, New Haven (1992)].   The production of the topology is one of several ways to model peptides with ideal geometry. To use the theoretical information of the reams (description of atoms in valence angle, contribution of covalent bonds). You. The following steps use the method of distance geometry. Positive measured by NMR The distance between the elementary distances and the theoretical distance associated with the primary structure (the length of the bond, The rigidity of the aromatic ring). A random series of distances between the approved distance intervals Sorting exists. These distances are then assigned to each atom in the molecule. To Cartesian coordinates. Apply an optimization process; it will affect the structure produced Minimizing the target function that represents the error with respect to the experimental distance observed It is in. As wide as possible as a result of random sorting of a set of distances No single structure is produced to explore any structural space, but Is produced. These structures satisfy some of the experimental distance constraints; However, imperfections still often exist in its covalent geometry, The energy is generally high. Then, molecular mechanics using stimulated annealing The adjustment of the structure according to It takes 20 picoseconds during the 50 ° K phase for 9 picoseconds. 0 Temperature rise to 0 ° K, then equilibration for 12 picoseconds, and 10 picoseconds During the 25 ° K step to 250 ° K. By stimulated annealing The second step of purifying the structure is performed at 1000 K for 50 picoseconds and The energy minimization is subsequently performed by a 500 Powell cycle. This In the step, the weight of the Van der Waals interaction is kept low. Until today Rapidly produce structures whose amplified parameters are compatible with NMR data (E.g., during the stimulated annealing step) The limits of energy have been suppressed). Therefore, the last step in this modeling is It is to minimize the potential energy of various structures provided. Example 2: Antigenicity of the peptide of SEQ ID NO: 2   The applicant determined the antigenic properties of the peptide by the ELISA test described below. Analyzed and performed using serum obtained from patients infected with HCV. Tested 1 For each of the 0 sera, additional wells containing no patient's , Used as an internal control. a) Protocol for ELISA test   The method involves the use of specific antibodies in serum or plasma from patients, and It is possible to indicate the presence of the corresponding antigen in the body.   Wells of a microtiter plate under the trade name "NUNC maxisorb" for 2 hours PH 9.6 carboxylic acid buffer solution containing the peptide (10 μg / ml) Saturate using 100 μl. Then empty the plate and add 0.05% Tw Wash with the help of wash buffer containing een20. The wells are 10% Saturation was performed using 100 μl of wash buffer supplemented with bovine serum (v / v). Incubate at 30 ° C for 30 minutes, then wash again as above. Analyzed The serum is diluted in an appropriate manner using a saturation buffer and then at 37 ° C. for 1 hour. Incubate the wells for a while. Wash the wells again. Then the saturated battery Conjugates (labeled using peroxidase) at a dilution of 1/1000 in fur Of goat IgG anti-human IgG). After 10 minutes, 50 μl of H_TwoSO_Four Block the reaction using and read the light intensity at 492 nm. b) Results   Table 1 below classifies the results of the ELISA tests together and shows the peroxidase reaction. Expressed as a subsequent light intensity (OD) reading at 492 nm. This table shows the Light intensity (OD) observed for tides is more common than for control wells The peptide is substantially higher, including substantially all anti-nucleocapsid antibodies. Shows strong reactivity with serum. Serum H662 to which the peptide did not react was 1 + Type, which is only slightly reactive to nucleocapsids. It is a serum that can be cited as an example.   The peptide does not contain an antibody directed against nucleocapsid "neg Finally, it should be noted that it does not cross-react with two sera called " .                                   Table 1 ^*Serum was classified according to its reactivity and given a number: eg RIBA 3 (sold by ORTHO DIAGNOSTIC) The serum with the number 4+ reacts strongly and the serum with the number 1+ reacts weakly. Example 3: NMR characterization of the three-dimensional structure of a peptide of the invention   Reference peptide previously identified by two amino acids for experimental reasons (SEQ ID NO: 2) synthesize a longer peptide called capsid (15-41), The peptide was then converted to 40% TFE / 60% H_TwoO, 0.1 M NaCl, 1 Example 1 by placing in 20 mM sodium phosphate at pH 5.9 in 0 mM sodium phosphate. Was characterized by NMR according to the methods and materials identified in .   FIG. 2 shows 2048 points in dimension t1 and 548 points in dimension t2. NOR of the peptide recorded using a mixing time of 250 ms for 12 points 3 shows an SY spectrum. At t1 and t2, at 4K and 2K, respectively. Zero filling was applied.   The filtration has a square sign type in both dimensions. This space Vectors are a more specific representative of the secondary structure of the considered peptide.   The spectrum according to FIG. 3 was recorded with a mixing time of 250 ms. P The correlation between the β proton of he24 and the γ and δ protons of Ile30 was shown. Has the characteristics of the quaternary structure defined in Example 4: Molecular modeling of the three-dimensional structure of peptide capsid (15-41)   Molecular modeling of the peptide capsid (15-41) was performed at 252 and 2 respectively. Performed using 68 stresses (Table 2 below). Produced for each peptide Of the 50 structures, 32 allowed structures are peptide capsids (15-4 1) was obtained.   The peptide capsid (15-41) model indicates that this peptide is actually Contains two helices, these two helices actually maintain perpendicular to each other To indicate that Therefore, the three-dimensional helix-loop-helix unit is Is found for   Table 2 shows that the inter-residue stress (i, i) is positively carried by the same amino acid residue. NMR stress on peptide capsid (15-41) corresponding to offspring A continuous stress (i, i + 1) will produce a proton that saturates two adjacent residues. Bind; what is called medium distance stress (i, i + 2, i + 4) Containing protons separated by most four residues of Less (i, i> i + 4) is the positive number of residues separated by more than four amino acids. Combine children.                                   Table 2

[Procedure for Amendment] Article 184-8, Paragraph 1 of the Patent Act [Date of Submission] June 15, 1999 (June 15, 1999) [Details of Amendment] Claims 1. Recognized by an antibody directed against a polypeptide comprising the N-terminal 2-45 amino acid sequence of the core protein or nucleocapsid protein (p21) of hepatitis C virus (HCV) or a variant of any of the above viruses A structural peptide, comprising a peptide sequence equivalent to SEQ ID NO: 2 or consisting of the peptide sequence of SEQ ID NO: 2, wherein the peptide sequence is a first peptide fragment having a secondary structure in the form of an α helix, α A second peptide fragment having a secondary structure in the form of a helix, and a third binding peptide fragment joining the two α helices, wherein the two α helices are substantially perpendicular to each other in space; N of the p21 protein considered to start at amino acid 1 or 2 Including the end or all of a protein or peptide compound consisting therewith, and the following sequence of SEQ ID NO: 11 A structural peptide characterized by excluding 2. The structural peptide according to claim 1, wherein the two α helices form an angle of 90 ° ± 10 °. 3. The structural peptide according to claim 1, wherein the third fragment is selected from a loop or an L-shape. 4. The structural peptide according to claim 1, wherein the peptide sequence equivalent to SEQ ID NO: 2 is selected from any one of the sequences SEQ ID NO: 3 to SEQ ID NO: 10. 5. The structural peptide according to any one of claims 1 to 4, comprising a peptide sequence equivalent to SEQ ID NO: 2. 6. A polypeptide comprising the N-terminal 2-45 amino acid sequence of the hepatitis C virus (HCV) core protein (p21), comprising the peptide according to any one of claims 1 to 5. An antigenic compound that is recognized by an antibody directed against it. 7. 6. The N-terminal 2-terminal of the core protein (p21) of hepatitis C virus (HCV) or a mutant of any of the above-mentioned viruses, comprising the peptide according to any one of claims 1 to 5. An immunogenic compound capable of inducing the production of antibodies directed against a polypeptide comprising a sequence of 45 amino acids. 8. A monoclonal or polyclonal antibody obtainable by in vivo or in vitro immunization of an animal using the immunogenic compound of claim 7. 9. Use of a peptide according to any one of claims 1 to 5, or a compound according to claim 6, for the detection and / or quantification of antibodies directed against HCV p21. 10. Use of the antibody according to claim 9 for detecting and / or quantifying the antigen of HCV or a mutant of any of the above viruses. 11. 6. A method according to any one of claims 1 to 5 for preparing a diagnostic, prophylactic or therapeutic composition capable of detecting, preventing or treating infection by HCV or a mutant of any of the above viruses. Use of the peptide according to claim 1, the compound according to claim 6 or 7, or the antibody according to claim 8. 12. A pharmaceutical composition comprising the peptide according to any one of claims 1 to 5, the compound according to claim 6 or 7, or the antibody according to claim 8. 13. A method for detecting and / or quantifying an antibody directed against a core protein (p21) of hepatitis C virus (HCV) in a sample, wherein the sample is a sample according to any one of claims 1 to 5. A method comprising contacting with a peptide and / or a compound according to claim 6 and detecting the formation of an immune complex between said antibody and said peptide or compound. 14． A method for detecting and / or quantifying an antigen of HCV or a mutant of said virus in a sample, said sample being contacted with an antibody according to claim 8, wherein said sample comprises an immune complex between said antigen and said antibody. A method comprising detecting formation. 15. A method for detecting and / or quantifying all or a part of RNA of hepatitis C virus in a sample, wherein the sample is the peptide according to any one of claims 1 to 5 and / or 6 or 7. A method for detecting the formation of a complex between the RNA and the peptide or the compound, wherein the method is contacted with the compound according to the above. 16. A complex comprising a peptide according to claims 1 to 5 and a molecular structure that binds specifically to said peptide, wherein said molecular structure is a peptide fragment, a nucleotide fragment and a chemical such as of the functionalized aromatic type. A conjugate characterized in that it is particularly selected from the target molecules. [Procedural amendment] [Submission date] September 7, 1999 (1999.9.7) [Content of amendment] In the description, page 2, lines 9 to 12, "Advantageously, ... The explanation of the terms is described below. ""Advantageously, the peptide sequence of the peptide of the invention is selected from any peptide sequence equivalent to SEQ ID NO: 2, such as SEQ ID NO: 3 to 10. Preferably, the peptide of the invention is SEQ ID NO: 1 Before describing the present invention in more detail and presenting its significance and applications, a description of the terms used in the present description and claims follows.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12Q 1/70 G01N 33/576 Z G01N 33/576 A61K 37/02 (81) Designated countries EP (AT, BE) , CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN) , ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, E , FI, GB, GE, GH, GM, GW, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, M G, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Ladaviere, Laurent France 69100 Vieux Vannes Rue Enard 43 (72) Inventor Lacu, Xavier France 69008 Lyon Rue de Montagny 92

Claims (1)

[Claims] 1. Core of hepatitis C virus (HCV) or a variant of any of the above viruses N-terminal 2-45 amino acid of protein or nucleocapsid protein (p21) A structure recognized by an antibody directed against a polypeptide comprising the amino acid sequence First peptide having a secondary structure in the form of an alpha helix Fragment, a second peptide fragment having a secondary structure in the form of an α-helix, And a third binding peptide fragment that binds the two α helices, Two α-helices are substantially perpendicular to each other in space, and the amino acid at position 1 Or including the N-terminus of the p21 protein which is considered to start at the amino acid at position 2 Or excluding all protein or peptide compounds consisting of Structural peptide 2. The two α-helices form an angle of 90 ° ± 10 °. The structural peptide according to claim 1. 3. 2. The method according to claim 1, wherein the third fragment is selected from a loop or an L-shape. The structural peptide according to any one of the preceding claims. 4. The structural peptide according to claim 1, comprising the quaternary structure. 5. Further, it is characterized by including any peptide sequence equivalent to SEQ ID NO: 2. A structural peptide according to any one of claims 1 to 4. 6. A peptide comprising the peptide according to any one of claims 1 to 5. N-terminal 2-45 of hepatitis C virus (HCV) core protein (p21) Recognized by antibodies directed against polypeptides containing amino acid sequences Antigenic compounds. 7. A peptide comprising the peptide according to any one of claims 1 to 5. Of hepatitis C virus (HCV) or a variant of any of the above viruses For the polypeptide containing the N-terminal 2-45 amino acid sequence of the protein (p21), An immunogenic compound capable of inducing the production of antibodies directed to the subject. 8. 8. In vivo immunization of an animal with the immunogenic compound of claim 7; Are monoclonal or polyclones obtainable by in vitro immunization Null antibody. 9. For the detection and / or quantification of antibodies directed against HCV p21, A peptide according to any one of claims 1 to 5, or a compound according to claim 6. Use of compounds. 10. Detecting antigens of HCV or a mutant of any of the above viruses and / or Use of the antibody according to claim 9 for quantification. 11. Detection and prevention of infection by HCV or a mutant of any of the above viruses Or prepare a diagnostic, prophylactic or therapeutic composition that can be treated The peptide according to any one of claims 1 to 5, Use of a compound according to claim 1 or an antibody according to claim 8. 12. The peptide according to any one of claims 1 to 5, wherein the peptide according to claim 6 or 7. A pharmaceutical composition comprising the compound of claim 8 or the antibody of claim 8. 13. Directed against hepatitis C virus (HCV) core protein (p21) A method for detecting and / or quantifying the antibody in a sample, wherein the sample is The peptide according to any one of claims 1 to 5 and / or the peptide according to claim 6. Contact with the above-mentioned compound, and obtain an immunological pattern between the antibody and the peptide or compound. A method comprising detecting the formation of coalescence. 14． Detecting the antigen of HCV or a mutant of the above virus in the sample and / or Is a method of quantification, wherein the sample is contacted with the antibody according to claim 8, A method comprising detecting the formation of an immune complex between said antigen and said antibody. 15. All or part of the hepatitis C virus RNA is detected and / or Or the method of quantification, wherein the sample is described in any one of claims 1 to 5. And / or a compound according to claim 6 or 7; Specifically, detecting the formation of a complex between the RNA and the peptide or compound. How to sign. 16. A peptide according to any one of claims 1 to 5, and the peptide described above. A complex comprising a differentially binding molecular structure, wherein the molecular structure is a peptide fragment From chemical molecules such as, nucleotide fragments and those of the functionalized aromatic type. A complex, wherein the complex is selected from the group consisting of: